Sarma Rajeev Kumar

Stress-Induced Accumulation of DcAOX1 and DcAOX2a Transcripts Coincides with Critical Time Point for Structural Biomass Prediction in Carrot Primary Cultures (Daucus carota L.).

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes’ were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.

Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves

The monoterpene indole alkaloids (MIAs) are generally derived from strictosidine, which is formed by condensation of the terpene moiety secologanin and the indole moiety tryptamine. There are conflicting reports on the limitation of either terpene or indole moiety in the production of MIAs in Catharanthus roseus cell cultures. Formation of geraniol by geraniol synthase (GES) is the first step in secologanin biosynthesis. In this study, feeding of C. roseus leaves with geraniol, but not tryptophan (precursor for tryptamine), increased the accumulation of the MIAs catharanthine and vindoline, indicating the limitation of geraniol in MIA biosynthesis. This was further validated by molecular and in planta characterization of C. roseus GES (CrGES). CrGES transcripts exhibited leaf and shoot specific expression and were induced by methyl jasmonate. Virus-induced gene silencing (VIGS) of CrGES significantly reduced the MIA content, which was restored to near-WT levels upon geraniol feeding. Moreover, over-expression of CrGES in C. roseus leaves increased MIA content. Further, CrGES exhibited correlation with MIA levels in leaves of different C. roseus cultivars and has significantly lower expression relative to other pathway genes. These results demonstrated that the transcriptional regulation of CrGES and thus, the in planta geraniol availability plays crucial role in MIA biosynthesis.

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree ( Murraya koenigii )

Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (−)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (−)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids.

Small RNAs: Master Regulators of Epigenetic Silencing in Plants

From fairly simple beginnings, research on epigenetic silencing in plants has revealed a highly complex epigenetic pathway. In the last two decades, several interesting phenomena associated with epigenetic regulation in plants were dissected giving insights into the biological significance of epigenetic marks and the role it plays in an organism’s life cycle by controlling different physiological processes like plant development, morphogenesis, reproduction, and stress response. Epigenetics refers to either heritable or reversible genetic modifications in DNA or histone proteins that maintain the nucleosome structure in a dynamic manner or those mediated by small RNAs (sRNAs) that in turn modulate gene expression. Plants are equipped with intricate regulatory mechanism to elicit highly sequence-specific chromatin-based gene silencing. Diverse classes of RNAs like small interfering RNA (siRNA), microRNAs (miRNAs), and long noncoding RNAs (lnc RNAs) have emerged as key regulators of gene expression along with several accessory proteins. sRNAs are widespread in various eukaryotes and are specifically involved in the maintenance of chromatin modifications in plants. These sRNAs regulate gene expression in different ways including post-transcriptional gene silencing (PTGS) in cytosol by targeting complementary transcripts for degradation, thereby repressing protein synthesis. In nucleus, sRNAs are responsible for transcriptional gene silencing (TGS) by directing epigenetic modifications like cytosine or histone methylation to homologous regions of the genome. This chapter gives an overview of the role of small RNAs in PTGS and TGS.

Genomics and Genetic Engineering in Phytoremediation of Arsenic

Arsenic (As) is one the extremely toxic metalloids that adversely affects health and hence it is categorized under group A human carcinogen. Generally, As-contaminated sites are not remediated due to high cost. Phytoremediation is the process of using plants to treat or clean up contaminated sites and it relies on natural ability of plants to extract, accumulate, or detoxify chemicals from water, soil, and air using energy from sunlight. Over the past several years, significant progress has been made to improve the effectiveness and efficiency of phytoremediation for removal of many hazardous metals from environment. Recent progress in understanding and identification of several genes involved in As uptake, transport, and metabolism in plants led to use of transgenic plants for remediation. Initial experiments of using transgenic plants as a tool to remove As were not promising; however the last decade witnessed a dramatic increase in the reports on the ability of plants to remove/detoxify As. Transgenic plants exploit the natural ability of plants, which rely on uptake of As by roots, transport through vascular system and leaf as a sink to concentrate. An array of genes from different sources including microbes, plants, and animals were successfully used to improve the ability of plants to tolerate, detoxify, and accumulate As. Transgenic plants containing specific genes converted toxic As to other forms that are less harmful. This review examines the recent advances in enhancing phytoremediation through transgenic approach for phytoremediation of As.

Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus

Catharanthus roseus is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in C. roseus. Here, to overcome this limitation and enhance MIAs levels in C. roseus, bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by Agrobacterium-mediated transformation. Both transient and stable overexpression of G(G)PPS and co-expression of G(G)PPS+GES significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic C. roseus plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in G(G)PPS but not in G(G)PPS+GES transgenic lines that correlated with transcript levels of peroxidase-1 (PRX1) involved in coupling of vindoline and catharanthine into 3′,4′-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T1) lines exhibited comparable transcript and metabolite levels to that of T0 lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in C. roseus at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in C. roseus through transgenic approaches.

Factors Affecting Genetic Transformation Efficiency in Sugarcane

Sugarcane (Saccharum officinarum) is an important cash crop cultivated across the world. Conventional breeding methods are used to cross different Saccharum spp. to develop sugarcane hybrids with high sucrose content and for other novel traits including increased tolerance to various biotic and abiotic stresses. Two major factors that limit conventional breeding method are that it is highly time consuming and difficulty in getting the desirable trait in the hybrid. These limitations can be overcome by genetic transformation method in which specific gene(s) are used to generate stable transgenic lines expressing specific trait. Compared to conventional breeding methods, generation of stable lines takes less time. In addition, complications associated with backcross and testcross during breeding program can be avoided. There are several reports since 1990s mentioning generation of transgenic sugarcane by different methods of transformation such as electroporation, particle bombardment method, and Agrobacterium-mediated transformation. Transient expression systems have also been developed in sugarcane. Nevertheless, all transformation methodologies have their own limitation which hinders the stable expression of the transformed gene. Here, we discuss about the complications and factors affecting efficiency of genetic transformation in sugarcane.

Plastome Engineering: Yesterday, Today, and Tomorrow

Plant transformation has made significant strides in last two decades with main focus on developing stress-tolerant crops and pharmaceutically important compounds for therapeutic purpose. There are many success stories describing the production of therapeutic proteins in large scale that are targeted to either nuclear or plastid genomes. The plastid genome (plastome) represents an attractive target for genetic engineering in crop plants. Transgenes integrated to plastome have several advantages like high expression levels, genes can be stacked in operons and genes integrated to plastome do not exhibit silencing mechanism. An additional advantage lies in the maternal inheritance of plastids in most plant species, which addresses the biosafety concerns related to transgenic plants. The plastid engineering usually results in alteration of several thousand plastid genome copies in a cell. In this chapter, the evolution of this technology with respect to the current state-of-the-art methods and the advantage of this technology over nuclear transformation are discussed. The recent advancement in plastome engineering and novel tools/methods developed to overcome potential limitations of chloroplast transformation are discussed in this chapter. Finally, future application of chloroplast engineering with a perspective for sugarcane plastome engineering is also briefed.